The specific and accurate attachment of amino acids onto tRNA molecules is a critical step in protein biosynthesis. RNA and amino acid recognition and discrimination by aminoacyl-tRNA synthetases play essential roles in this process. Due to the high degree of specificity between tRNAs and aminoacyl-tRNA synthetases, these are ideal molecules for investigating the principles that govern protein-RNA interactions. The twenty synthetases have been partitioned into two classes of ten members each. Over the last decade, structural information on synthetases and tRNAs from both classes has provided insights into synthetase-substrate interactions and mechanisms of tRNA and amino acid discrimination. There are common features that appear to be class-specific, such as the structure of the catalytic domain, the mode of ATP binding, and the site of initial amino acid attachment. In contrast, there is considerable variation in the mode of amino acid and tRNA recognition. Thus, it is important to study in detail each of the twenty aminoacyl-tRNA synthetases, as each will provide new insights into molecular interactions responsible for the accurate translation of the genetic code and improve our understanding of protein-RNA interactions in general. Moreover, the aminoacyl-tRNA synthetases are an essential family of enzymes offering novel targets for new antibiotic development. The recent explosion of sequence and structural information emphasizes the enormous diversity of these enzymes. In particular, comparison of prokaryotic synthetases with the corresponding eukaryotic enzymes including those from human (all of which are now sequenced) reveals many species-specific distinctions that may, in principle, be selectively targeted for inhibitor development. Class II aminoacyl-tRNA synthetases have been the subject of research in the Musier-Forsyth lab for the past seven years and this effort will be extended and expanded in the proposed work. The specific aims of this project are: (1) To explore species- specific differences in tRNA recognition by prolyl-tRNA synthetases (ProRS), (2) to explore the function of the large insertion domain present in the "prokaryotic-like" ProRS group in aminoacyl-adenylate ("pre-transfer") and aminoacyl-tRNA ("post- transfer") editing, (3) to explore the relationship between specific sequence elements, RNA conformation, and class II aminoacyl-tRNA synthetase recognition of acceptor stem-derived RNA microhelices, and (4) to explore the mechanism of communication between key anticodon elements important for aminoacylation by class IIb synthetases and the tRNA acceptor stem.